Sex disparity in viral load, inflammation and liver damage in transgenic mice carrying full hepatitis B virus genome with the W4P mutation in the preS1 region

AIM To study sex disparity in susceptibility to hepatocellular carcinoma(HCC), we created a transgenic mouse model that expressed the full hepatitis B virus(HBV) genome with the W4P mutation.METHODS Transgenic mice were generated by transferring the p HY92-1.1 x-HBV-full genome plasmid(genotype A2) into C57 Bl/6 N mice. We compared serum levels of hepatitis B surface antigen(HBs Ag), interleukin(IL)-6, and the liver enzymes alanine aminotransferase(ALT) and aspartate transaminase(AST), as well as liver histopathological features in male and female transgenic(W4PTG) mice and in nontransgenic littermates of 10 mo of age. RESULTS W4PTG males exhibited more pronounced hepatomegaly, significantly increased granule generation in liver tissue, elevated HBs Ag expression in the liver and serum, and higher serum ALT and IL-6 levels compared to W4PTG females or littermate control groups. CONCLUSION Together, our data indicate that the W4 P mutation in pre S1 may contribute to sex disparity in susceptibility to HCC by causing increased HBV virion replication and enhanced IL-6-mediated inflammation in male individuals. Additionally, our transgenic mouse model that expresses full HBV genome with the W4 P mutation in pre S1 could be effectively used for the studies of the progression of liver diseases, including HCC.

Analysis of hepatitis B virus preS1 variability and prevalence of the rs2296651 polymorphism in a Spanish population

AIM To determine the variability/conservation of the domain of hepatitis B virus(HBV) pre S1 region that interacts with sodium-taurocholate cotransporting polypeptide(hereafter, NTCP-interacting domain) and the prevalence of the rs2296651 polymorphism(S267 F, NTCP variant) in a Spanish population. METHODS Serum samples from 246 individuals were included and divided into 3 groups: patients with chronic HBV infection(CHB)(n = 41, 73% Caucasians), patients with resolved HBV infection(n = 100, 100% Caucasians) and an HBV-uninfected control group(n = 105, 100% Caucasians). Variability/conservation of the amino acid(aa) sequences of the NTCPinteracting domain,(aa 2-48 in viral genotype D) and a highly conserved pre S1 domain associated with virion morphogenesis(aa 92-103 in viral genotype D) were analyzed by next-generation sequencing and compared in 18 CHB patients with viremia > 4 log IU/mL. The rs2296651 polymorphism was determined in all individuals in all 3 groups using an in-house real-time PCR melting curve analysis.RESULTS The HBV pre S1 NTCP-interacting domain showed a high degree of conservation among the examined viral genomes especially between aa 9 and 21(in the genotype D consensus sequence). As compared with the virion morphogenesis domain, the NTCPinteracting domain had a smaller proportion of HBV genotype-unrelated changes comprising > 1% of the quasispecies(25.5% vs 31.8%), but a larger proportion of genotype-associated viral polymorphisms(34% vs 27.3%), according to consensus sequences from Gen Bank patterns of HBV genotypes A to H. Variation/conservation in both domains depended on viral genotype, with genotype C being the most highly conserved and genotype E the most variable(limited finding, only 2 genotype E included). Of note, proline residues were highly conserved in both domains, and serine residues showed changes only to threonine or tyrosine in the virion morphogenesis domain. The rs2296651 polymorphism was not detected in any participant.CONCLUSION In our CHB population, the NTCP-interacting domain was highly conserved, particularly the proline residues and essential amino acids related with the NTCP interaction, and the prevalence of rs2296651 was low/null.

Expression and immunoactivity of chimeric particulate antigens of receptor binding site-core antigen of hepatitis B virus

AIM: To improve the immunogenicity of receptor binding site of hepatitis B virus (HBV) on preS1 antigen using HBV core antigen as an immuno-carrier. METHODS: One to 6 tandem copies of HBV preS1 (21-47) fragment were inserted into HBcAg at the sites of aa 78 and 82, and expressed in E.coli. ELISA, Western blot and animal immunization were used to analyze the antigenicity and immmunogenicity of purified particulate antigens. The ability to capture HBV by antibodies elicited by chimeric particles was detected with immuno-capture PCR. RESULTS: Recombinant antigens CI, CII, CIII carrying 1-3 copies of HBV preSl (21-47) individually could form virus-like particles (VLPs), similar to HBcAg in morphology. But recombinant antigens carrying 4-6 copies of HBV preSl (21-47) were poorly expressed in E.coli. Chimeric antigens were lacking of immunoreactivity with anti-HBc monoclonal antibodies (McAbs), but still reserved good immunoreactivity with anti-HBe McAbs. CI, CII, CIII could strongly react with anti-preS1 McAb, suggesting that preS1 (21-47) fragment was well exposed on the surface of chimeric VLPs. Three chimeric VLP antigens (CI, CII and CIII) could stimulate mice to produce high-level antibody responses, and their immunogenicity was stronger than non-particulate antigen 21-47*6, containing 6 copies of preS1 (21-47). Mouse antibodies to CI, CII and CIII were able to capture HBV virions in immuno-capture PCR assay in vitro. CONCLUSION: Chimeric particulate antigens of receptor binding site-core antigen of HBV can elicit strong antibody responses to preS1. They have a potential to be developed into prophylactic or therapeutic vaccines against HBV infection.

Cloning and expression of the preS1 gene of hepatitis B virus in yeast cells

Objective: To investigate the complex functions of HBV preS1 protein, we constructed HBV preS1 gene expression vector and expressed it in yeast cells. Methods: Polymerase chain reaction (PCR) was per- formed to amplify the gene of HBV preS1 from the plasmid pCP10 containing the whole DNA fragment of HBV ayw subtype as template and the PCR prod- uct was cloned into the pGEM-T vector for sequen- cing. After being identified, the HBV preSl gene was cut from the pGEM-T vector by EcoR I and Pst I restriction enzymes, and cloned into yeast expres- sive plasmid pGBKT7 to constructe pGBKT7-preS1 recombinant expressive plasmid. This plasmid was transformed into yeast cell AH109 and expressed in it. The yeast protein was isolated and analyzed with sodium dodecyl suifate-polyacrylamide gel electro- phoresis(SDS-PAGE) and Western blotting. Results: The HBV preS1 gene was amplified success- fully and identified by DNA sequencing. The PCR products were coincided completely with the reported sequence. The digested fragments were cloned into the pGBKT7 vector and transformed into yeast cell AH109. The results of SDS-PAGE and Western blot- ting assay showed: (1) The HBV preS1 protein was expressed and existed in yeast cells; (2) The molecu- lar weight of the expression product was about 30 000 D. Conclusion: The HBV preS1 gene was successfully cloned and expressed in yeast cells.

HBV DNA level and antigen concentration in evaluating liver damage of patients with chronic hepatitis B

OBJECTIVE: To investigate the correlations between HBV DNA levels and viral antigen concentrations in patients with chronic hepatitis B and their significance in clinical practice. METHODS: The HBV DNA levels and serological markers of 118 patients with chronic hepatitis B and 87 patients with liver cirrhosis who had not been treated with antiviral drugs were determined as well as the other parameters relevant to liver function. RESULTS: The HBV DNA levels of the patients with chronic hepatitis anti cirrhosis were expressed as geometric mean±SD, 3.83×10~6±1.34 copies/ml anti 6.98×10~5±1.29 copies/ml, and their HBeAg concentrations expressed as the luminescent values rate of sample to control (s/co) were 35.40±1.26 and 4.05±1.28, respectively. The HBV DNA levels in HBeAg positive group were significantly higher than those in HBeAg negative group (P<0.0001). The correlation coefficient between HBV DNA level and HBeAg or HBsAg concentration was only O. 273 anti -0.12. During the recovery of hepatic function, the reduction of ALT or AST in patients with high viral content was significantly lower than that in patients with low viral content. No correlation was observed between HBV DNA and ALT levels. CONCLUSION: There are significant correlations between HBV DNA level anti HBeAg concentration, but the coefficient is lower. HBV DNA level is not significantly related to ALT, but it could affect the recovery of liver function.

Hepatitis B virus pre S1 deletion is related to viral replication increase and disease progression

AIM:To investigate the clinical implications of hepatitis B virus(HBV) pre S1 deletion.METHODS:We developed a fluorescence resonance energy transfer-based real-time polymerase chain reaction(RT-PCR) that can detect four genotypes(wild type, 15-bp, 18-bp and 21-bp deletion).The PCR method was used in two cohorts of Korean chronic HBV subjects with genotype C infections.Cohort Ⅰ included 292 chronic HBV subjects randomly selected from Cheju National University Hospital(Jeju, South Korea) or Seoul National University Hospital(Seoul, South Korea), and cohort Ⅱ included 90 consecutive chronic HBV carriers recruited from Konkuk University Hospital(Seoul, South Korea); the cohort Ⅱ patients did not have hepatocellular carcinoma or liver cirrhosis.RESULTS:The method proposed in this study identified 341 of 382 samples(89.3%).Deletion variants were identified in 100(29.3%) of the 341 detected samples.In both cohorts, the subjects with deletions had a significantly higher Hepatitis B virus e antigen(HBe Ag)-positive seroprevalence [cohort Ⅰ, wild(51.0%) vs deletion(75.0%), P < 0.001; cohort Ⅱ, wild(69.2%) vs deletion(92.9%), P = 0.002] and higher HBV DNA levels [cohort Ⅰ, wild(797.7 pg/m L) vs deletion(1678.9 pg/m L), P = 0.013; cohort Ⅱ, wild(8.3 × 108 copies/m L) vs deletion(2.2 × 109 copies/m L), P = 0.049], compared to subjects with wild type HBV.CONCLUSION:HBV genotype C pre S1 deletion may affect disease progression in chronic HBV subjects through an extended duration of HBe Ag seropositive status and increased HBV replications.

mi R-29a promotes hepatitis B virus replication and expression by targeting SMARCE1 in hepatoma carcinoma

AIM To investigate the functional role and underlying molecular mechanism of mi R-29 a in hepatitis B virus(HBV) expression and replication.METHODS The levels of mi R-29 a and SMARCE1 in HBV-infected Hep G2.2.15 cells were measured by quantitative real-time PCR and western blot analysis. HBV DNA replication was measured by quantitative PCR and Southern blot analysis. The relative levels of hepatitis B surface antigen and hepatitis B e antigen were detected by enzyme-linked immunosorbent assay. The Cell Counting Kit-8(CCK-8) was used to detect the viability of Hep G2.2.15 cells. The relationship between mi R-29 a and SMARCE1 were identified by target prediction and luciferase reporter analysis.RESULTS mi R-29 a promoted HBV replication and expression, w h i le S MA R C E 1 r e p r e s s e d H B V r e p lic a t io n a n d expression. Cell viability detection indicated that mi R-29 a transfection had no adverse effect on the host cells. Moreover, SMARCE1 was identified and validated to be a functional target of mi R-29 a. Furthermore, restored expression of SMARCE1 could relieve the increased HBV replication and expression caused by mi R-29 a overexpression.CONCLUSION mi R-29 a promotes HBV replication and expression through regulating SMARCE1. As a potential regulator of HBV replication and expression, mi R-29 a could be a promising therapeutic target for patients with HBV infection.

Generation of high affinity human single-hain antibody against PreSl of hepatitis B virus from immune phage-display antibody library

BACKGROUND: A single-chain antibody ( ScFv) phage display library was created by cloning antigen-binding re- gions of VH (variable domain) and VL gene repertoires as fusion proteins with a minor coat protein of filamentous phage, from which high affinity completely humanized ScFv against PreS1 of hepatitis B virus could be screened and characterized. METHODS: A combinatorial library of phage-display hu- man ScFv genes, which were derived from peripheral blood lymphocytes immunized by peptide PreS1 in vitro, was constructed. The library contained 7 × 108 clones. RESULTS: After 3 rounds panning, a high affinity (K = 10-7-10-8 mol/L) ScFv specific to PreS1 was obtained. Sequence analysis showed that the VH belonged to the VH4 family and Vλ to Vλ4. CONCLUSIONS: The described ScFv may provide a more satisfactory therapy. This application further illustrates that the method of in vitro antigen stimulation is expeditious for the source of human immune antibody library.

HBeAg阴性乙肝患者血清前S1抗原检测分析

目的探讨HBeAg阴性乙肝患者血清前S1抗原检测的意义及应用价值。方法用ELISA方法对314例慢性乙型肝炎患者血清进行前S1抗原的测定并对其HBV-DNA含量进行分析研究。分析170例HBeAg阴性乙肝患者和144例HBeAg阳性的乙肝患者血清中HBV-DNA的含量。结果在170例血清HBeAg阴性患者中,60%为PreS1阳性,其阳性率显著低于144例HBeAg阳性患者,PreS1阳性检出率为76%(χ2=9.55,P<0.005)。在212例PreS1阳性标本中,HBV-DNA阳性检出率为94%,其病毒载量(log)为5.78±1.94,HBV-DNA阳性率和HBV-DNA病毒载量均显著高于PreS1阴性患者(102例)HBV-DNA阳性率为82%(χ2=11.45,P<0.005)和病毒载量(log)4.35±2.33,(t=5.36,P=0.000 1)。在HBeAg阴性患者中,90%的PreS1(+)者为HBV-DNA阳性,其病毒载量为(log)4.89±2.00,显著高于PreS1阴性患者的HBV-DNA阳性检出率(75%)(χ2=7.52,P<0.01),和病毒载量(log)37.7±2.46(t=3.13,P=0.002)。结论乙肝病毒前S1抗原的检测能敏感的反映乙肝病毒的复制,当HBeAg阴性时PreS1阳性可预测血清HBV-DNA的状态,PerS1检测具有重要的临床价值。

HBeAg阴性及HBeAg阳性慢性乙型肝炎患者的临床特征比较

目的分析HBeAg阴性与HBeAg阳性慢性乙型肝炎患者临床特征差异。方法对290例慢性乙型肝炎患者的住院病历进行临床特征采集,分析HBeAg阴性与HBeAg阳性慢性乙型肝炎患者年龄、性别、家族史、病程、肝功能、HBV DNA定量、HBsAg定量、HBV的表面抗原蛋白前S1抗原(Pre-S1Ag)等指标的差异。结果 HBeAg阴性组年龄高于HBeAg阳性组,HBeAg阴性组病程长于HBeAg阳性组,HBeAg阴性组谷草转氨酶低于HBeAg阳性组,HBeAg阴性组HBsAg定量低于HBeAg阳性组,差异有显著性(P<0.05);45.91%的HBeAg阴性组患者HBV DNA<10~4copies/ml,59.39%的HBeAg阳性组患者HBV DNA病毒载量>10~6copies/ml,差异有显著性(P<0.05);HBeAg阴性组Pre-S1A异阳性率(50.81%)低于HBeAg阳性组(75.98%),差异有显著性(P<0.05)。结论虽然HBeAg阳性组年龄轻、病程短,但是其HBV病毒载量、HBsAg定量、Pre-S1Ag阳性率更高,Pre-S1Ag、乙肝表面抗原定量、HBV DNA能很好地反映病毒在体内复制及疾病进展情况。

HBeAg阴性慢性乙肝患者血清前S1抗原与HBV DNA的关系

目的通过分析HBV Pre-S1抗原与HBV DNA的关系,探讨HBeAg阴性慢性乙肝患者血清前S1抗原检测的临床价值。方法采用ELISA法、荧光定量PCR法及生化检测270例HBeAg阴性慢性乙肝患者及50例健康对照血清Pre-S1抗原、HBV DNA及肝功能,并对检查结果进行相关性分析。结果 270例HBeAg阴性血清中,Pre-S1抗原和HBV DNA阳性率分别为39.6%和41.9%,Pre-S1抗原与HBV DNA存在相关性(χ2=187.0,P<0.01);Pre-S1抗原阳性组AST、ALT、TBIL、γ-GT均高于阴性组(P<0.01);当HBV DNA拷贝数的对数值>2.7时,Pre-S1抗原诊断敏感度85.8%,特异度93.6%,准确性90.4%,阳性预测值90.7%,阳性似然比13.4,优势比89.1。结论 HBV Pre-S1抗原与HBV DNA、肝功能均有较好相关性,在HBeAg阴性慢性乙肝患者中,它能够敏感、准确地反映HBV的复制和肝功能状况,可作为乙肝病毒复制的一项新传染性指标。

乙型肝炎病毒前S1抗原与HBV DNA及HBeAg关系

目的 探讨乙型肝炎病毒 (HBV)前S1抗原 (PreS1Ag)与HBVDNA和HBeAg之间的关系 ,以评价其在乙肝诊疗中的作用。方法 用ELISA法对 2 2 5例HBeAg阳性标本进行PreS1Ag和HBV血清标志物的检测 ,用PCR-ELISA法测定HBVDNA。结果 PreS1Ag在HBVDNA和HBsAg阳性组织中的检出率均明显高于相应的阴性组(均为P <0 0 1)。HBeAg阴性组中PreS1Ag的检出率为 4 3 0 % ,PreS1Ag与HBVDNA的符合率为 70 6 % ,与HBeAg的符合率为 6 8 4 %。结论 PreS1Ag与HBVDNA、HBeAg呈良好的相关性。作为反映HBV感染和复制的指标 ,PreS1Ag较HBeAg更敏感。

HBV感染者血清中HBeAg，HBeAb与Pre—S1抗原的相关性研究

目的探讨HBV感染者HBeAg，HBeAb与Pre-S1抗原的关系及其临床价值。方法收集2012年1月～3月HBV感染者血清1080例，采用酶联免疫吸附法（ELISA）检测HBsAg，HBsAb，HBeAg，HBeAb，HBcAb及Pre-S1抗原，应用SPSS17．0统计软件分析HBeAg，HBeAb与Pre-S1抗原的相关性。结果在1080例HBV感染者中，HBeAg阳性240例，HBeAg阴性840例，HBeAg阳性者和阴性者Pre-S1抗原阳性率分别为91．25％和70．12％，HBeAg阳性者Pre-S1抗原阳性率明显高于HBeAg阴性者，两者比较差异有统计学意义（x^2=44．24，P〈o．05）。在840例HBeAg阴性者中，HBeAb阳性者636例，HBeAb阴性者204例，HBeAb阳性者和阴性者Pre-S1抗原阳性率分别为65．72％和83．82％，HBeAb阳性者中Pre-S1抗原阳性率明显低于HBeAb阴性者，两者比较差异有统计学意义（x^2-24．15，P〈0．05）。结论血清HBeAg，HBeAb与Pre-S1抗原存在相关性。Pre-S1抗原阳性提示HBV复制活跃，传染性较强，Pre-S1抗原阴性可作为HBV被清除和感染趋于恢复，病情趋向好转的标志。

HBeAg阴性慢性乙型肝炎和非活动期HBsAg携带状态的Pre-S1抗原阳性率研究

目的探讨Pre-S1抗原对HBeAg阴性慢性乙型肝炎发生的诊断意义。方法选择HBeAg阴性慢性乙型肝炎病例和非活动期HBsAg携带状态病例各45例作为研究组,并以HBeAg阳性慢性乙型肝炎和免疫耐受期病例各20例分别作为其对照组。应用酶联免疫法(ELISA)测HBsAg,HBsAb,HBeAg,HBeAb,HBcAb,Pre-S1抗原,应用聚合酶链反应(PCR)检测HBV-DNA。结果 HBeAg阴性慢性乙型肝炎组与非活动期HBsAg携带状态组Pre-S1抗原阳性率有统计学差异(χ2=4.121,P=0.035)。HBeAg阴性慢性乙型肝炎组与HBeAg阳性慢性乙型肝炎组Pre-S1抗原阳性率无统计学差异(χ2=2.921,P=0.083)。非活动期HBsAg携带状态组与免疫耐受组Pre-S1抗原检测阳性率有显著性差异(χ2=6.573,P=0.009)。结论 Pre-S1抗原可作为观察非活动期HBsAg携带状态是否稳定的监测指标,如出现Pre-S1抗原阳性提示非活动期HBsAg携带状态易发展为HBeAg阴性慢性乙型肝炎。

血清中HBV DNA含量、Pre-S1蛋白及HBeAg之间的关系研究

目的通过分析HBV DNA含量、Pre-S1蛋白及HBeAg之间的关系,探讨三者在反映乙肝病毒复制中的价值及临床意义。方法采用酶联免疫吸附试验(ELISA)检测HBsAg阳性标本的Pre-S1及HBe-Ag,酶标仪检测其吸光度值,实时荧光定量聚合酶联反应(PCR)检测HBV DNA含量。结果在HBV DNA阳性或阴性标本中,Pre-S1阳性检出率明显高于HBeAg(P=0.005),但Pre-S1的吸光度值在两者中无差异,而HBeAg在HBV DNA阳性标本中的吸光度(S/C0=21.167±9.597)高于在阴性标本中的值(S/C0=2.915±1.683),两者比较P=0.006;在HBeAg阳性标本中,HBV DNA和Pre-S1的阳性检出率无差异,而在阴性标本中Pre-S1的阳性检出率明显高于HBV DNA检出率,并且HBV DNA含量和Pre-S1吸光度值两者差异有统计学意义(P<0.05);在Pre-S1阳性标本中,HBV DNA阳性率高于HBeAg的阳性率,而且HBV DNA含量也高于在阴性标本中含量(P=0.022)。结论 HBV DNA含量、Pre-S1蛋白及HBeAg在血清中表达意义的侧重点可能不同,在实际工作中应联合检测,才能较全面地反映病毒在患者体内的真实情况,为临床治疗提供更加客观可靠的实验数据。

慢性乙肝治疗路线图和HBV DNA、HBeAg、HBsAg定量的监测意义

慢性乙型肝炎病毒感染呈世界分布并且引起不良结局,是极为严重的健康问题。慢性乙型肝炎治疗最重要的目标是长期抑制病毒以改善预后,防止出现肝硬化、肝衰竭和肝癌。目前,抗病毒药物主要有干扰素和口服核苷(酸)类似物,核苷(酸)类似物的缺点主要在于长期治疗中出现耐药。路线图概念被用于监测耐药预测疗效。HBV DNA定量水平被作为路线图较好的监测指标。由于血清转换率及清除率过低,HBeAg、HBsAg定量检测不适合用于路线图监测,但对于使用聚乙二醇干扰素的慢性乙肝患者,可作为预测治疗应答的指标。

乙型肝炎病毒S1抗原检测及其与HBeAg的关系探讨

目的探讨乙型肝炎病毒(HBV)前S1抗原与HBeAg及HBV在体内复制和感染的存在关系。方法采用ELISA法对280例乙肝肝炎病毒感染者中HBeAg阳性患者血清进行前S1抗原检测。结果乙型肝炎病毒(HBV)前S1抗原在血清学表达上与HBeAg的阳性符合率高达91.43％。结论乙型肝炎病毒(HBV)前S1抗原与HBeAg平行存在,是乙型肝炎病毒在体内存在的复制和感染的可靠指标。

乙型肝炎病毒前S1抗原与HBV DNA、HBeAg的相关性分析——附178例检测分析

目的:探讨HBV前S1抗原(pre-S1antigen,Pre-S1Ag)与HBV DNA、HBeAg的相关性,并评价Pre-S1Ag的临床应用价值。方法:对178例慢性乙型病毒性肝炎(慢乙肝)患者采用ELISA法检测Pre-S1Ag、HBeAg,采用荧光定量PCR法检测HBV DNA,比较Pre-S1Ag、HBeAg与HBV DNA的诊断符合率,并根据HBV DNA载量分为低复制组、中等复制组及高复制组,分析HBV DNA载量与Pre-S1Ag阳性率的相关性。结果:178例的HBVDNA均为阳性,其中Pre-S1Ag阳性144例(80.9%),Pre-S1Ag阴性34例(19.1%);HBeAg阳性112例(62.9%),HBeAg阴性66例(37.1%)。Pre-S1Ag、HBeAg与HBV DNA的诊断符合率分别为80.9%、62.9%,上述两诊断符合率比较差异有统计学意义(P<0.05)。112例HBeAg阳性慢乙肝者中,Pre-S1Ag的阳性率80.4%;66例HBeAg阴性慢乙肝者中,Pre-S1Ag阳性率82%。HBV高复制组的Pre-S1Ag阳性率(93%)高于中等复制组(80%)和低复制组(64%),且HBV DNA载量与Pre-S1Ag的阳性率呈正相关(r=0.293,P<0.01)。结论:Pre-S1Ag与HBV DNA有较好的相关性,在无法开展HBV DNA检测的基层医院,对于HBeAg阴性慢乙肝患者应增加检测Pre-S1Ag,以提高检测的准确度。

HBcAg基因和PreS1(1～65)肽基因真核重组载体的构建和鉴定

目的：构建并鉴定包含人乙型肝炎病毒（HBV）HBcAg基因和PreS1（1～65）肽基因的真核重组载体。方法：采用PCR法扩增HBcAg基因，并通过基因突变在79～80位氨基酸处生成一个NheⅠ酶切位点，然后将这段基因连接到真核表达载体pIRES2-EGFP启动子下游的EcoRⅠ和BamHⅠ之间。同时PCR扩增PreS1第1～65氨基酸基因，并在其两端分别引入NheⅠ酶切位点，通过酶切、连接，将这段基因插入到HBcAg基因的NheⅠ酶切位点上。将构建的重组载体pIREScS1转化大肠杆菌DH5α，分别用上述内切酶双酶切及DNA测序鉴定重组质粒。结果：酶切鉴定示，插入片段大小均与预计相符。测序结果与已知序列结果一致。结论：成功构建了HBcAg基因和PreS1（1～65）肽基因的真核重组载体。